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1.
Cell Rep ; 36(8): 109570, 2021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34390647

RESUMO

The rapid development of mRNA-based vaccines against the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) led to the design of accelerated vaccination schedules that have been extremely effective in naive individuals. While a two-dose immunization regimen with the BNT162b2 vaccine has been demonstrated to provide a 95% efficacy in naive individuals, the effects of the second vaccine dose in individuals who have previously recovered from natural SARS-CoV-2 infection has not been investigated in detail. In this study, we characterize SARS-CoV-2 spike-specific humoral and cellular immunity in naive and previously infected individuals during and after two doses of BNT162b2 vaccination. Our results demonstrate that, while the second dose increases both the humoral and cellular immunity in naive individuals, COVID-19 recovered individuals reach their peak of immunity after the first dose. These results suggests that a second dose, according to the current standard regimen of vaccination, may be not necessary in individuals previously infected with SARS-CoV-2.


Assuntos
COVID-19/prevenção & controle , Linfócitos T/imunologia , Vacinas Sintéticas/administração & dosagem , Anticorpos Antivirais/sangue , Ligante de CD40/metabolismo , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Vacinas contra COVID-19/administração & dosagem , Vacinas contra COVID-19/química , Vacinas contra COVID-19/imunologia , Humanos , Imunidade Celular , Imunidade Humoral , Imunoglobulina G/sangue , Interferon gama/metabolismo , Interleucina-2/metabolismo , Peptídeos/imunologia , SARS-CoV-2/isolamento & purificação , SARS-CoV-2/metabolismo , Glicoproteína da Espícula de Coronavírus/imunologia , Linfócitos T/citologia , Linfócitos T/metabolismo , Vacinação , Vacinas Sintéticas/imunologia
2.
Nat Commun ; 12(1): 4785, 2021 08 09.
Artigo em Inglês | MEDLINE | ID: mdl-34373459

RESUMO

The implementation of applied engineering principles to create synthetic biological systems promises to revolutionize medicine, but application of fundamentally redesigned organisms has thus far not impacted practical drug development. Here we utilize an engineered microbial organism with a six-letter semi-synthetic DNA code to generate a library of site-specific, click chemistry compatible amino acid substitutions in the human cytokine IL-2. Targeted covalent modification of IL-2 variants with PEG polymers and screening identifies compounds with distinct IL-2 receptor specificities and improved pharmacological properties. One variant, termed THOR-707, selectively engages the IL-2 receptor beta/gamma complex without engagement of the IL-2 receptor alpha. In mice, administration of THOR-707 results in large-scale activation and amplification of CD8+ T cells and NK cells, without Treg expansion characteristic of IL-2. In syngeneic B16-F10 tumor-bearing mice, THOR-707 enhances drug accumulation in the tumor tissue, stimulates tumor-infiltrating CD8+ T and NK cells, and leads to a dose-dependent reduction of tumor growth. These results support further characterization of the immune modulatory, anti-tumor properties of THOR-707 and represent a fundamental advance in the application of synthetic biology to medicine, leveraging engineered semi-synthetic organisms as cellular factories to facilitate discovery and production of differentiated classes of chemically modified biologics.


Assuntos
Antineoplásicos/uso terapêutico , Interleucina-2/química , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Animais , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/imunologia , Descoberta de Drogas , Engenharia Genética , Humanos , Interleucina-2/genética , Subunidade alfa de Receptor de Interleucina-2 , Células Matadoras Naturais/efeitos dos fármacos , Células Matadoras Naturais/imunologia , Linfócitos/efeitos dos fármacos , Camundongos , Biologia Sintética
3.
J Clin Exp Hematop ; 61(3): 145-151, 2021 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-34334531

RESUMO

We established an IL-2 and IL-4 (IL2/4) - dependent adult T-cell leukemia/lymphoma (ATLL) cell line (YG-PLL) by adding poly-L-lysine (PLL) to the culture medium. YG-PLL originates from lymphoma cells and contains a defective HTLV-I proviral genome. Although YG-PLL cannot survive without IL-2/4, the follicular dendritic cell (FDC)-like cell line HK expressing OX40-ligand gene (OX40L+HK) inhibited their death in the presence of soluble neutral polymers. After the prevention of cell death, YG-PLL proliferated on OX40L+HK without IL2/4 in the presence of two kinds of positively or negatively charged polymers. In particular, dermatan sulfate and poly-L-histidine supported growth for more than 4 months. Therefore, the original lymphoma cells proliferated transiently in the presence of IL2/4, and their growth arrest was inhibited by the addition of PLL. Furthermore, YG-PLL lost IL2/4 dependency by the following 3-step procedure: preculture with IL2/4 and neutral polymers, 3-day culture with neutral polymer on OX40L+HK to inhibit cell death, and co-culture with OX40L+HK in the presence of the positively and negatively charged polymers. The extracellular environment made by soluble polymers plays a role in the growth of ATLL in vitro.


Assuntos
Linhagem Celular Tumoral , Dermatan Sulfato/farmacologia , Histidina/farmacologia , Interleucina-2/metabolismo , Interleucina-4/metabolismo , Leucemia-Linfoma de Células T do Adulto/etiologia , Leucemia-Linfoma de Células T do Adulto/metabolismo , Ligante OX40/metabolismo , Células Dendríticas Foliculares/imunologia , Células Dendríticas Foliculares/metabolismo , Expressão Gênica , Humanos , Leucemia-Linfoma de Células T do Adulto/patologia , Ligante OX40/genética
4.
Theranostics ; 11(14): 6668-6681, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093846

RESUMO

Background: Newly emerging cancer immunotherapy has led to significant progress in cancer treatment; however, its efficacy is limited in solid tumors since the majority of them are "cold" tumors. Oncolytic viruses, especially when properly armed, can directly target tumor cells and indirectly modulate the tumor microenvironment (TME), resulting in "hot" tumors. These viruses can be applied as a cancer immunotherapy approach either alone or in combination with other cancer immunotherapies. Cytokines are good candidates to arm oncolytic viruses. IL-23, an IL-12 cytokine family member, plays many roles in cancer immunity. Here, we used oncolytic vaccinia viruses to deliver IL-23 variants into the tumor bed and explored their activity in cancer treatment on multiple tumor models. Methods: Oncolytic vaccinia viruses expressing IL-23 variants were generated by homologue recombination. The characteristics of these viruses were in vitro evaluated by RT-qPCR, ELISA, flow cytometry and cytotoxicity assay. The antitumor effects of these viruses were evaluated on multiple tumor models in vivo and the mechanisms were investigated by RT-qPCR and flow cytometry. Results: IL-23 prolonged viral persistence, probably mediated by up-regulated IL-10. The sustainable IL-23 expression and viral oncolysis elevated the expression of Th1 chemokines and antitumor factors such as IFN-γ, TNF-α, Perforin, IL-2, Granzyme B and activated T cells in the TME, transforming the TME to be more conducive to antitumor immunity. This leads to a systemic antitumor effect which is dependent on CD8+ and CD4+ T cells and IFN-γ. Oncolytic vaccinia viruses could not deliver stable IL-23A to the tumor, attributed to the elevated tristetraprolin which can destabilize the IL-23A mRNA after the viral treatment; whereas vaccinia viruses could deliver membrane-bound IL-23 to elicit a potent antitumor effect which might avoid the possible toxicity normally associated with systemic cytokine exposure. Conclusion: Either secreted or membrane-bound IL-23-armed vaccinia virus can induce potent antitumor effects and IL-23 is a candidate cytokine to arm oncolytic viruses for cancer immunotherapy.


Assuntos
Adenocarcinoma/terapia , Neoplasias do Colo/terapia , Imunoterapia/métodos , Interleucina-23/farmacologia , Vírus Oncolíticos/genética , Microambiente Tumoral/imunologia , Vírus Vaccinia/genética , Adenocarcinoma/imunologia , Adenocarcinoma/virologia , Animais , Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Neoplasias do Colo/imunologia , Neoplasias do Colo/virologia , Modelos Animais de Doenças , Feminino , Vetores Genéticos , Granzimas/metabolismo , Humanos , Interferon gama/metabolismo , Interleucina-12/metabolismo , Interleucina-2/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Vírus Oncolíticos/metabolismo , Perforina/metabolismo , Microambiente Tumoral/genética , Fator de Necrose Tumoral alfa/metabolismo , Vírus Vaccinia/metabolismo
5.
Front Immunol ; 12: 648348, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34079542

RESUMO

Dendritic cells (DCs) in peripheral tissues may have a unique role to regulate innate and adaptive immune responses to antigens that enter the tissues. Peritoneal cavity is the body compartment surrounding various tissues and organs and housing diverse immune cells. Here, we investigated the specialized features of classical DC (cDC) subsets following the intraperitoneal injection of a model antigen ovalbumin (OVA). Peritoneal cDC1s were superior to cDC2s in activating OVA-specific CD8 T cells, while both cDCs were similar in stimulating OVA-specific CD4 T cells. Each peritoneal cDC subset differentially regulated the homing properties of CD8 T cells. CD8 T cells stimulated by cDC1s displayed a higher level of lung-homing receptor CCR4, whereas those stimulated by cDC2s prominently expressed various homing receptors including gut-homing molecules CCR9 and α4ß7. Also, we found that cDC1s played a dominating role over cDC2s in controlling the overall gene expression of CD8 T cells. Soluble factor(s) emanating from CD8 T cells stimulated by peritoneal cDC1s were responsible for mediating this dominance of cDC1s, and we identified IL-2 as a soluble factor regulating the global gene expression of T cells. Collectively, our study indicates that different peritoneal cDC subsets effectively diversify T cell responses by altering the level of cytokines, such as IL-2, in the milieu.


Assuntos
Linfócitos T CD4-Positivos/imunologia , Linfócitos T CD8-Positivos/imunologia , Comunicação Celular/genética , Células Dendríticas/imunologia , Regulação da Expressão Gênica , Interleucina-2/metabolismo , Cavidade Peritoneal/citologia , Animais , Apresentação do Antígeno/efeitos dos fármacos , Antígenos/administração & dosagem , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/efeitos dos fármacos , Células Cultivadas , Células Dendríticas/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Interleucina-2/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Ovalbumina/administração & dosagem , Receptores CCR/metabolismo , Receptores CCR4/metabolismo
6.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070449

RESUMO

Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells that are responsible for immunosuppression in tumor microenvironment. Here we report the impact of mucin 1 (MUC1), a transmembrane glycoprotein, on proliferation and functional activity of MDSCs. To determine the role of MUC1 in MDSC phenotype, we analyzed MDSCs derived from wild type (WT) and MUC1-knockout (MUC1KO) mice bearing syngeneic pancreatic (KCKO) or breast (C57MG) tumors. We observed enhanced tumor growth of pancreatic and breast tumors in the MUC1KO mice compared to the WT mice. Enhanced tumor growth in the MUC1KO mice was associated with increased numbers of suppressive MDSCs and T regulatory (Tregs) cells in the tumor microenvironment. Compared to the WT host, MUC1KO host showed higher levels of iNOS, ARG1, and TGF-ß, thus promoting proliferation of MDSCs with an immature and immune suppressive phenotype. When co-cultured with effector T cells, MDSCs from MUC1KO mice led to higher repression of IL-2 and IFN-γ production by T cells as compared to MDSCs from WT mice. Lastly, MDSCs from MUC1KO mice showed higher levels of c-Myc and activated pSTAT3 as compared to MDSCs from WT mice, suggesting increased survival, proliferation, and prevention of maturation of MDSCs in the MUC1KO host. We report diminished T cell function in the KO versus WT mice. In summary, the data suggest that MUC1 may regulate signaling pathways that are critical to maintain the immunosuppressive properties of MDSCs.


Assuntos
Neoplasias da Mama/metabolismo , Mucina-1/metabolismo , Células Supressoras Mieloides/imunologia , Neoplasias Pancreáticas/metabolismo , Linfócitos T Reguladores/imunologia , Microambiente Tumoral/imunologia , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , Técnicas de Cocultura , Modelos Animais de Doenças , Feminino , Interferon gama/metabolismo , Interleucina-2/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucina-1/genética , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/imunologia , Baço/citologia , Baço/metabolismo , Fator de Crescimento Transformador beta/sangue , Microambiente Tumoral/genética
7.
J Immunol ; 206(10): 2322-2337, 2021 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-33931484

RESUMO

The costimulatory receptor CD28 synergizes with the TCR to promote IL-2 production, cell survival, and proliferation; yet the obligatory interdependence of TCR and CD28 signaling is not well understood. Upon TCR stimulation, Gads, a Grb2-family adaptor, bridges the interaction of two additional adaptors, LAT and SLP-76, to form a TCR-induced effector signaling complex. SLP-76 binds the Tec-family tyrosine kinase, Itk, which phosphorylates SLP-76 Y173 and PLC-γ1 Y783. In this study, we identified TCR-inducible, Itk-mediated phosphorylation of Gads Y45 in a human T cell line and in mouse primary T cells. Y45 is found within the N-terminal SH3 domain of Gads, an evolutionarily conserved domain with no known signaling function. Gads Y45 phosphorylation depended on the interaction of Gads with SLP-76 and on the dimerization-dependent binding of Gads to phospho-LAT. We provide evidence that Itk acts through SLP-76 and Gads to promote the TCR/CD28-induced activation of the RE/AP transcriptional element from the IL-2 promoter. Two Itk-related features of SLP-76, Y173 and a proline-rich Itk SH3 binding motif on SLP-76, were dispensable for activation of NFAT but selectively required for the TCR/CD28-induced increase in cytoplasmic and nuclear c-Rel and consequent RE/AP activation. We provide evidence that unphosphorylated, monomeric Gads mediates an RE/AP-directed inhibitory activity that is mitigated upon Gads dimerization and Y45 phosphorylation. This study illuminates a new, to our knowledge, regulatory module, in which TCR-induced, Itk-mediated phosphorylation sites on SLP-76 and Gads control the transcriptional response to TCR/CD28 costimulation, thus enforcing the obligatory interdependence of the TCR and CD28 signaling pathways.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/deficiência , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antígenos CD28/metabolismo , Fosfoproteínas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Dimerização , Vetores Genéticos , Humanos , Interleucina-2/metabolismo , Células Jurkat , Camundongos , Camundongos Endogâmicos BALB C , Fosfoproteínas/genética , Fosforilação/genética , Ligação Proteica , Transfecção
8.
Science ; 372(6543)2021 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-33986151

RESUMO

Reactive oxygen species (ROS) increase in activated T cells because of metabolic activity induced to support T cell proliferation and differentiation. We show that these ROS trigger an oxidative stress response that leads to translation repression. This response is countered by Schlafen 2 (SLFN2), which directly binds transfer RNAs (tRNAs) to protect them from cleavage by the ribonuclease angiogenin. T cell-specific SLFN2 deficiency results in the accumulation of tRNA fragments, which inhibit translation and promote stress-granule formation. Interleukin-2 receptor ß (IL-2Rß) and IL-2Rγ fail to be translationally up-regulated after T cell receptor stimulation, rendering SLFN2-deficient T cells insensitive to interleukin-2's mitogenic effects. SLFN2 confers resistance against the ROS-mediated translation-inhibitory effects of oxidative stress normally induced by T cell activation, permitting the robust protein synthesis necessary for T cell expansion and immunity.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Imunidade Celular , Estresse Oxidativo , RNA de Transferência/metabolismo , Linfócitos T/imunologia , Animais , Proteínas de Ciclo Celular/genética , Proliferação de Células , Feminino , Deleção de Genes , Infecções por Herpesviridae/imunologia , Subunidade gama Comum de Receptores de Interleucina/genética , Subunidade gama Comum de Receptores de Interleucina/metabolismo , Interleucina-2/metabolismo , Subunidade beta de Receptor de Interleucina-2/genética , Subunidade beta de Receptor de Interleucina-2/metabolismo , Ativação Linfocitária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Muromegalovirus , Ligação Proteica , Biossíntese de Proteínas , Espécies Reativas de Oxigênio/metabolismo , Receptores de Antígenos de Linfócitos T/imunologia , Receptores de Antígenos de Linfócitos T/metabolismo , Ribonuclease Pancreático/genética , Ribonuclease Pancreático/metabolismo , Transdução de Sinais
9.
PLoS One ; 16(5): e0250278, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34014928

RESUMO

Alzheimer's disease (AD) is a heterogeneous disease and exhibits diverse clinical presentations and disease progression. Some pathological and anatomical subtypes have been proposed. However, these subtypes provide a limited mechanistic understanding for AD. Leveraging gene expression data of 222 AD patients from The Religious Orders Study and Memory and Aging Project (ROSMAP) Study, we identified two AD molecular subtypes (synaptic type and inflammatory type) using consensus non-negative matrix factorization (NMF). Synaptic type is characterized by disrupted synaptic vesicle priming and recycling and synaptic plasticity. Inflammatory type is characterized by disrupted IL2, interferon alpha and gamma pathways. The two AD molecular subtypes were validated using independent data from Gene Expression Omnibus. We further demonstrated that the two molecular subtypes are associated with APOE genotypes, with synaptic type more prevalent in AD patients with E3E4 genotype and inflammatory type more prevalent in AD patients with E3E3 genotype (p = 0.031). In addition, two molecular subtypes are differentially represented in male and female AD, with synaptic type more prevalent in male and inflammatory type in female patients (p = 0.051). Identification of AD molecular subtypes has potential in facilitating disease mechanism understanding, clinical trial design, drug discovery, and precision medicine for AD.


Assuntos
Doença de Alzheimer/classificação , Genótipo , Redes e Vias Metabólicas , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/epidemiologia , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteínas E/genética , Feminino , Frequência do Gene , Humanos , Interferons/metabolismo , Interleucina-2/metabolismo , Masculino , Pessoa de Meia-Idade , Modelos Estatísticos , Prevalência , Fatores Sexuais
10.
Biochem Biophys Res Commun ; 556: 53-58, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33839414

RESUMO

Interleukin 15 receptor (IL-15R) is a transmembrane signalling protein consisting of 3 subsets: α, ß (IL-15Rß), and γ (γc). IL-2 and IL-15 share the signalling domains IL-15Rß and γc, although they bind to intrinsic α-subsets and non-signalling domains. Additionally, IL-2 and IL-15 play different roles; therefore, there have been many observations of the dynamic behaviours of IL-15R, which are linked to physiological functions. For more practical discrimination between IL-2 and IL-15, a study was designed and carried out in which α-subsets were removed and a cytoplasmic inhibitor was applied to create a simplified environment in which secondary signalling molecules were reduced. We also applied a new measurement method, diffracted X-ray blinking (DXB), to achieve higher accuracy (<0.01 Å). The dynamics of IL-2 binding (confined motion, max range = 0.71 Å) and IL-15 binding (normal motion) in live natural killer cells were different. We also confirmed. that DXB was a suitable method to quantitatively evaluate the transmembrane protein dynamics of inner/outer live cell membranes by labeling the extracellular domain since the measurements were dependent on the cytosolic environment.


Assuntos
Células Matadoras Naturais/citologia , Células Matadoras Naturais/metabolismo , Receptores de Interleucina-15/análise , Receptores de Interleucina-15/metabolismo , Difração de Raios X/métodos , Sobrevivência Celular , Difusão , Humanos , Hidroxibenzoatos , Interleucina-15/metabolismo , Interleucina-2/metabolismo , Simulação de Dinâmica Molecular , Nitrofuranos , Domínios Proteicos , Especificidade por Substrato
11.
Front Immunol ; 12: 657768, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33854514

RESUMO

Autoimmune diseases affect roughly 5-10% of the total population, with women affected more than men. The standard treatment for autoimmune or autoinflammatory diseases had long been immunosuppressive agents until the advent of immunomodulatory biologic drugs, which aimed at blocking inflammatory mediators, including proinflammatory cytokines. At the frontier of these biologic drugs are TNF-α blockers. These therapies inhibit the proinflammatory action of TNF-α in common autoimmune diseases such as rheumatoid arthritis, psoriasis, ulcerative colitis, and Crohn's disease. TNF-α blockade quickly became the "standard of care" for these autoimmune diseases due to their effectiveness in controlling disease and decreasing patient's adverse risk profiles compared to broad-spectrum immunosuppressive agents. However, anti-TNF-α therapies have limitations, including known adverse safety risk, loss of therapeutic efficacy due to drug resistance, and lack of efficacy in numerous autoimmune diseases, including multiple sclerosis. The next wave of truly transformative therapeutics should aspire to provide a cure by selectively suppressing pathogenic autoantigen-specific immune responses while leaving the rest of the immune system intact to control infectious diseases and malignancies. In this review, we will focus on three main areas of active research in immune tolerance. First, tolerogenic vaccines aiming at robust, lasting autoantigen-specific immune tolerance. Second, T cell therapies using Tregs (either polyclonal, antigen-specific, or genetically engineered to express chimeric antigen receptors) to establish active dominant immune tolerance or T cells (engineered to express chimeric antigen receptors) to delete pathogenic immune cells. Third, IL-2 therapies aiming at expanding immunosuppressive regulatory T cells in vivo.


Assuntos
Tolerância Imunológica , Imunomodulação , Animais , Autoantígenos/imunologia , Doenças Autoimunes/etiologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/terapia , Terapia Baseada em Transplante de Células e Tecidos , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Humanos , Tolerância Imunológica/efeitos dos fármacos , Fatores Imunológicos , Imunomodulação/efeitos dos fármacos , Imunoterapia/métodos , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Interleucina-2/uso terapêutico , Linfócitos T/imunologia , Linfócitos T/metabolismo , Vacinas/administração & dosagem , Vacinas/imunologia
12.
Nat Commun ; 12(1): 2029, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33795689

RESUMO

Mucosal-associated invariant T (MAIT) cells have important functions in immune responses against pathogens and in diseases, but mechanisms controlling MAIT cell development and effector lineage differentiation remain unclear. Here, we report that IL-2/IL-15 receptor ß chain and inducible costimulatory (ICOS) not only serve as lineage-specific markers for IFN-γ-producing MAIT1 and IL-17A-producing MAIT17 cells, but are also important for their differentiation, respectively. Both IL-2 and IL-15 induce mTOR activation, T-bet upregulation, and subsequent MAIT cell, especially MAIT1 cell, expansion. By contrast, IL-1ß induces more MAIT17 than MAIT1 cells, while IL-23 alone promotes MAIT17 cell proliferation and survival, but synergizes with IL-1ß to induce strong MAIT17 cell expansion in an mTOR-dependent manner. Moreover, mTOR is dispensable for early MAIT cell development, yet pivotal for MAIT cell effector differentiation. Our results thus show that mTORC2 integrates signals from ICOS and IL-1ßR/IL-23R to exert a crucial role for MAIT17 differentiation, while the IL-2/IL-15R-mTORC1-T-bet axis ensures MAIT1 differentiation.


Assuntos
Citocinas/imunologia , Proteína Coestimuladora de Linfócitos T Induzíveis/imunologia , Ativação Linfocitária/imunologia , Alvo Mecanístico do Complexo 1 de Rapamicina/imunologia , Alvo Mecanístico do Complexo 2 de Rapamicina/imunologia , Células T Invariantes Associadas à Mucosa/imunologia , Animais , Diferenciação Celular/imunologia , Células Cultivadas , Citocinas/metabolismo , Humanos , Proteína Coestimuladora de Linfócitos T Induzíveis/metabolismo , Interleucina-15/imunologia , Interleucina-15/metabolismo , Interleucina-2/imunologia , Interleucina-2/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina/metabolismo , Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células T Invariantes Associadas à Mucosa/citologia , Células T Invariantes Associadas à Mucosa/metabolismo , Transdução de Sinais/imunologia , Serina-Treonina Quinases TOR/imunologia , Serina-Treonina Quinases TOR/metabolismo
13.
Aging (Albany NY) ; 13(5): 7199-7210, 2021 02 26.
Artigo em Inglês | MEDLINE | ID: mdl-33653969

RESUMO

Chimeric antigen receptor (CAR) T cells target specific tumor antigens and lyse tumor cells in an MHC-independent manner. However, the efficacy of CAR-T cell and other cancer immunotherapies is limited by the expression of immune-checkpoint molecules such as programmed death-ligand 1 (PD-L1) on tumor cells, which binds to PD-1 receptors on T cells leading to T cell inactivation and immune escape. Here, we incorporated a PD-L1-targeted single-chain variable fragment (scFv) fusion protein sequence into a CAR vector to generate human anti-PD-L1-CAR-T cells (aPDL1-CART cells) targeting the PD-L1 antigen. Unlike control T cells, aPDL1-CART cells significantly halted the expansion and reduced the viability of co-cultured leukemia cells (Raji, CD46, and K562) overexpressing PD-L1, and this effect was paralleled by increased secretion of IL-2 and IFN-γ. The antitumor efficacy of aPDL1-CART cells was also evaluated in vivo by co-injecting control T cells or aPDL1-CART cells along with PDL1-CA46 cells to generate subcutaneous xenografts in NCG mice. Whereas large tumors developed in mice inoculated with PDL1-CA46 cells alone or together with control T cells, no tumor formation was detected in xenografts containing aPDL1-CART cells. Our data suggest that immune checkpoint-targeted CAR-T cells may be useful for controlling and eradicating immune-refractory hematological malignancies.


Assuntos
Antígeno B7-H1/imunologia , Imunoterapia Adotiva/métodos , Leucemia Experimental/terapia , Animais , Linhagem Celular Tumoral , Humanos , Interferon gama/metabolismo , Interleucina-2/metabolismo , Leucemia Experimental/imunologia , Camundongos , Transplante de Neoplasias
14.
Front Immunol ; 12: 628063, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33717150

RESUMO

Tumor infiltrating lymphocytes (TIL) therapy was shown to provide durable objective response in patients with metastatic melanoma. As a fundamental first step to bring TIL therapy to clinical use, identification of patients whose tumors yield optimal numbers of reactive TIL is indispensable. We have previously shown that expansion of tumor reactive TIL from primary bladder tumors and lymph node metastases is feasible. Here, we performed TIL harvesting from additional surgical specimens (additional 31 primary tumors and 10 lymph nodes) to generate a heterogenous cohort of 53 patients with bladder cancer (BC) to evaluate the tumor characteristics that lead to tumor-reactive TIL expansion. Among a total of 53 patients, overall TIL growth from tumor samples were 37/53 (69.8%) and overall anti-tumor reactive TIL were 26/35 (74.3%). Mixed urothelial carcinoma is associated with higher anti-tumor reactivity of expanded TIL than pure urothelial carcinoma (89.5% vs. 56.3%, p=0.049). The anti-tumor reactivity of expanded TIL from primary tumors previously treated with BCG immunotherapy were lower (33.3% vs. 82.6%, p=0.027) although T-cell phenotype (CD3+, CD4+, CD8+, and CD56+) was similar regardless prior of BCG therapy. Addition of agonistic 4-1BB antibody in culture media with IL-2 improved the number of expanded TIL from primary tumors previously treated with BCG immunotherapy. There was no significant difference between basal and luminal subtype tumors in terms of viable and reactive TIL growth. Our study demonstrates that TIL expansion is feasible across all BC patients and BC subtypes, and we suggest that TIL therapy can be a reasonable treatment strategy for various manifestations of BC.


Assuntos
Vacinas Anticâncer/imunologia , Imunoterapia Adotiva/métodos , Linfócitos do Interstício Tumoral/fisiologia , Neoplasias da Bexiga Urinária/imunologia , Urotélio/patologia , Idoso , Proliferação de Células , Células Cultivadas , Estudos de Coortes , Feminino , Humanos , Interleucina-2/metabolismo , Metástase Linfática , Ativação Linfocitária , Masculino , Pessoa de Meia-Idade , Mycobacterium bovis , Membro 9 da Superfamília de Receptores de Fatores de Necrose Tumoral/agonistas , Neoplasias da Bexiga Urinária/terapia
15.
Melanoma Res ; 31(2): 162-172, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33661190

RESUMO

BACKGROUND: IL-2 inducible kinase (ITK) is highly expressed in metastatic melanomas and its inhibition suppresses melanoma cell proliferation. We hypothesize that ibrutinib has a direct antitumor effect in melanoma cell lines and that treatment of metastatic melanomas with ibrutinib induces antitumor responses. METHODS: We assessed the ibrutinib effect on melanoma cell proliferation, apoptosis, and motility. Patients with metastatic melanoma refractory to PD-1 and MAPK inhibitors (if BRAFV600-mutant) were treated with ibrutinib, 840 mg PO QD, as part of a phase II clinical trial (clinicaltrials.gov NCT02581930). RESULTS: Melanoma cell lines frequently express ITK, YES1, and EGFR. Ibrutinib suppressed cell motility and proliferation in most cell lines. Eighteen patients (13 male; median age 63.5 years, range 37-82; 12 with ipilimumab resistance) were enrolled. The most frequent side effects were fatigue (61%), anorexia (50%), hyponatremia (28%), nausea, and vomiting (22% each). No antitumor responses were seen. At a median follow-up of 6 months (0.3-35.8 months), the median progression-free survival was 1.3 months (range 0.2-5.5 months). Fifteen patients were discontinued from the study due to progression, and 14 patients had died from metastatic melanoma. All archived tumors expressed ITK, 41% had no expression of p16 and PTEN, and 61% had absent tumor-infiltrating lymphocytes (TILs). Ibrutinib significantly suppressed proliferating (Ki67+) CD19+ peripheral blood mononuclear cells and had no significant effect on other lymphocyte subsets. CONCLUSION: Ibrutinib did not induce any meaningful clinical benefit. ITK expression may not be clinically relevant. Treatment-refractory metastatic melanomas have other fundamental defects (i.e. absent PTEN and p16 expression, absent TILs) that may contribute to an adverse prognosis.


Assuntos
Adenina/análogos & derivados , Interleucina-2/metabolismo , Melanoma/tratamento farmacológico , Piperidinas/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Adenina/farmacologia , Adenina/uso terapêutico , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Piperidinas/farmacologia
16.
J Pharmacol Sci ; 145(4): 319-326, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33712283

RESUMO

Cannabinoid receptors are a potential target for anti-inflammatory and pain therapeutics. There are two subtypes, CB1 and CB2, and Δ9-tetrahydrocannabinol activates both of them, providing an analgesic effect but also psychoactive side effects. The psychoactive side effects are considered to be caused by activation of CB1, but not CB2. ABK5 is a CB2 subtype selective agonist that has a very different structure from known cannabinoid receptor agonists. Here, we report anti-inflammatory effects of ABK5 using the T-cell line Jurkat cells, and antinociceptive effect in an inflammatory pain model in rats. Production of the cytokines IL-2 and TNF-α was measured in stimulated Jurkat cells and MOLT-4 cells, and CXCL12-mediated chemotaxis of Jurkat cells was evaluated by a transwell migration assay. Anti-inflammatory and antinociceptive effects of ABK5 were also evaluated in a hindpaw CFA model in rats. ABK5 significantly decreased production of IL-2 and TNF-α measured as both mRNA and protein levels, and reduced chemotaxis towards CXCL12. It also attenuated edema and increased mechanical threshold in the hindpaw of CFA-treated rats. These results suggest that ABK5 is a good lead compound for the development of potential anti-inflammatory and analgesic agents.


Assuntos
Analgésicos , Anti-Inflamatórios , Dor/tratamento farmacológico , Receptor CB2 de Canabinoide/agonistas , Animais , Quimiocina CXCL12 , Quimiotaxia/efeitos dos fármacos , Modelos Animais de Doenças , Feminino , Humanos , Mediadores da Inflamação/metabolismo , Interleucina-2/metabolismo , Células Jurkat , Ratos Sprague-Dawley , Fator de Necrose Tumoral alfa/metabolismo
17.
J Tradit Chin Med ; 41(1): 36-43, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-33522195

RESUMO

OBJECTIVE: To investigate the efficacy of the extract from Yiyuan Yiliu Tang (, YYYLT) on human lung adenocarcinoma cells A549 and human hepatoma cells Bel7402. METHODS: The cancer cell lines were treated with various concentrations (0, 100, 200, 300, and 400 µg/mL) of the crude water extract of YYYLT and then cell viability, toxicity, cytokine secretion, and cell cycle/apoptosis were determined by MTT assay, LDH assay, and flow cytometry, respectively. RESULTS: The extract from YYYLT significantly suppressed the proliferation of the cancer cell lines and the release of interleukin-2 and tumor necrosis factor-α in a dose-dependent manner. The extract also promoted apoptosis, caused cell cycle arrest at G0/G1 phase, and increased the expression of caspase-3, B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X proteins. CONCLUSION: The extract from YYYLT might be a potential treatment for human lung and liver cancers.


Assuntos
Adenocarcinoma de Pulmão/tratamento farmacológico , Antineoplásicos Fitogênicos/farmacologia , Medicamentos de Ervas Chinesas/farmacologia , Neoplasias Pulmonares/tratamento farmacológico , Adenocarcinoma de Pulmão/genética , Adenocarcinoma de Pulmão/metabolismo , Adenocarcinoma de Pulmão/fisiopatologia , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Interleucina-2/genética , Interleucina-2/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/fisiopatologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/metabolismo
18.
Life Sci Alliance ; 4(4)2021 04.
Artigo em Inglês | MEDLINE | ID: mdl-33593878

RESUMO

IL-15 priming of NK cells is a broadly accepted concept, but the dynamics and underlying molecular mechanisms remain poorly understood. We show that as little as 5 min of IL-15 treatment in vitro, followed by removal of excess cytokines, results in a long-lasting, but reversible, augmentation of NK cell responsiveness upon activating receptor cross-linking. In contrast to long-term stimulation, improved NK cell function after short-term IL-15 priming was not associated with enhanced metabolism but was based on the increased steady-state phosphorylation level of signalling molecules downstream of activating receptors. Inhibition of JAK3 eliminated this priming effect, suggesting a cross talk between the IL-15 receptor and ITAM-dependent activating receptors. Increased signalling molecule phosphorylation levels, calcium flux, and IFN-γ secretion lasted for up to 3 h after IL-15 stimulation before returning to baseline. We conclude that IL-15 rapidly and reversibly primes NK cell function by modulating activating receptor signalling. Our findings suggest a mechanism by which NK cell reactivity can potentially be maintained in vivo based on only brief encounters with IL-15 trans-presenting cells.


Assuntos
Metabolismo Energético , Interleucina-15/metabolismo , Células Matadoras Naturais/imunologia , Células Matadoras Naturais/metabolismo , Transdução de Sinais , Animais , Biomarcadores , Citocinas/metabolismo , Imunofenotipagem , Interferon gama/metabolismo , Interleucina-15/farmacologia , Interleucina-2/metabolismo , Interleucina-2/farmacologia , Células Matadoras Naturais/efeitos dos fármacos , Ativação Linfocitária , Camundongos , Fosforilação , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
19.
Eur J Immunol ; 51(6): 1461-1472, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33548071

RESUMO

Blocking the mevalonate pathway for cholesterol reduction by using statin may have adverse effects including statin-induced colitis. Moreover, one of the predisposing factors for colitis is an imbalanced CD4+ T cell, which can be observed on the complete deletion of HMG-CoA reductase (HMGCR), a target of statins. In this study, we inquired geranylgeranyl pyrophosphate (GGPP) is responsible for maintaining the T-cell homeostasis. Following dextran sulfate sodium (DSS)-induced colitis, simvastatin increased the severity of disease, while cotreatment with GGPP, but not with cholesterol, reversed the disease magnitude. GGPP ameliorated DSS-induced colitis by increasing Treg cells. GGPP amplified Treg differentiation through increased IL-2/STAT 5 signaling. GGPP prenylated Ras protein, a prerequisite for extracellular signal-regulated kinase (ERK) pathway activation, leading to increased IL-2 production. Higher simvastatin dose increased the severity of colitis. GGPP ameliorated simvastatin-increased colitis by increasing Treg cells. Treg cells, which have the capacity to suppress inflammatory T cells and were generated through IL-2/STAT5 signaling, increased IL-2 production through prenylation and activation of the Ras/ERK pathway.


Assuntos
Anticolesterolemiantes/administração & dosagem , Linfócitos T CD4-Positivos/imunologia , Colite/imunologia , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/imunologia , Doenças Inflamatórias Intestinais/imunologia , Interleucina-2/metabolismo , Fosfatos de Poli-Isoprenil/uso terapêutico , Sinvastatina/administração & dosagem , Linfócitos T Reguladores/imunologia , Animais , Anticolesterolemiantes/efeitos adversos , Diferenciação Celular , Células Cultivadas , Colite/etiologia , Sulfato de Dextrana , Modelos Animais de Doenças , Homeostase , Humanos , Hidroximetilglutaril-CoA Redutases/genética , Ativação Linfocitária , Ácido Mevalônico/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Transdução de Sinais , Sinvastatina/efeitos adversos
20.
J Infect Dis ; 223(9): 1555-1563, 2021 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-33556959

RESUMO

Repeated infections with endemic human coronaviruses (hCoV) are thought to reflect lack of long-lasting protective immunity. We evaluated circulating human CD4 T cells collected prior to 2020 for reactivity towards hCoV spike proteins, probing for the ability to produce interferon-γ, interleukin-2, or granzyme B. We found robust reactivity to spike-derived epitopes, comparable to influenza, but highly variable abundance and functional potential across subjects, depending on age and viral antigen specificity. To explore potential of these memory cells to be recruited in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, we examined the subjects for cross-reactive recognition of epitopes from SARS-CoV-2 nucleocapsid, membrane/envelope, and spike. Functional potential of these cross-reactive CD4 T cells was highly variable; nucleocapsid-specific CD4 T cells but not spike-reactive cells showed exceptionally high levels of granzyme production upon stimulation. These results are considered in light of recruitment of hCoV-reactive cells into responses to SARS-CoV infections or vaccinations.


Assuntos
Linfócitos T CD4-Positivos/virologia , COVID-19/imunologia , Infecções por Coronavirus/imunologia , Epitopos de Linfócito T/imunologia , SARS-CoV-2/imunologia , Adulto , Idoso , Proteínas do Envelope de Coronavírus/imunologia , Proteínas M de Coronavírus/imunologia , Proteínas do Nucleocapsídeo de Coronavírus/imunologia , Reações Cruzadas , Granzimas/metabolismo , Humanos , Memória Imunológica , Interferon gama/metabolismo , Interleucina-2/metabolismo , Pessoa de Meia-Idade , Glicoproteína da Espícula de Coronavírus/imunologia
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